Contrasting small molecules, proteins are not only defined by their chemical formula. Specifically, while the identity of a small molecule can be often confirmed by mass spectrometric methods, conformational richness in proteins is critical for their function. Even small conformational changes that can be triggered by differences in pH can completely revert the enzymatic properties of a protein, as exemplified by the protease and ligase activity of legumain (Dail E. et al., “Structure and mechanism of an aspartimide-dependent Peptide ligase in human legumain”, Angew. Chem. Int. Ed. Engl. (2015), 54(1), 2917-21).
Given the intimate relation of protein structure and function, the exact knowledge of variations in protein structure is of utmost importance in particular for proteins that are therapeutically or diagnostically used. Lack-of-function changes may render a given therapeutic protein less active, which often can be compensated by an adjusted dosage; gain-of-function changes are often causing severe side effects. Typical gain-of-function changes are immune reactions which may lead to the generation of antibodies. These antibodies may neutralize the therapeutic agent, acting as inhibitors. Even worse, the immune reactions may mount to a severe inflammatory response, e.g. to a “cytokine storm”. In case of such an immune reaction, all related protein therapies may become inapplicable due to the cross-reactivity of the immune reaction. Furthermore, endogenous proteins that are homologous to the therapeutic agent are likely to be recognized by the triggered immune response as well, with possibly disastrous consequences.
In consequence, there is an increasing need in the art for detecting conformational changes in proteins and identifying proteins differing from other proteins only by their conformation, even though they may have sequence identity. In particular, it becomes of utmost importance to gain information about the protein structure and conformation of a protein of interest even from complex protein samples, which requires highly sensitive analytical methods. Further, the detection and identification of proteins differing from a reference protein only by minor conformational changes or posttranslational modifications is of utmost importance in the production and quality management of biosimilar products. In particular, the comparison of a similar biotherapeutic product and its reference biotherapeutic product plays a pivotal role in the assessment for overall biosimilarity.
Mass spectrometry is the work horse of protein analytics in academia and industry alike. Technological improvements allow the analysis of ever more complex protein samples and particularly extend the accessible mass range of the protein samples. Both top-down and bottom-up are typically used for protein identification. However, by the nature of this approach, conformational information on the target protein is hardly accessible. Thus, mass spectrometry is only of limited use when trying to detect small heterogeneities, such as small conformational differences, between proteins. Limited and indirect conformational information can only be obtained by laborious hydrogen-deuterium exchange experiments or the identification of disulphide bonds.
Mass spectrometric analysis is often complemented by an array of spectroscopic analytical techniques, including circular dichroism (CD), Fourier-Transform Infrared spectroscopy (FT-IR), NMR, or more specialized techniques like electron spin resonance or Mössbauer spectroscopy. These spectroscopic techniques may provide conformational information on the target protein, albeit at relatively low resolution. X-ray diffraction may be employed to obtain high resolution (atomic) information on protein structures. However, crystallization is challenging and not always successful, in particular with complex or heterogeneous protein samples. Furthermore, crystallization represents a selection process which bears the intrinsic risk to exclude protein species/conformations which may be functionally important.
New approaches have been reported that employ limited proteolysis (LiP) as a tool to sample the conformation of a target protein in solution.
Feng et al., “Global analysis of protein structural changes in complex proteomes”, Nature Biotechnology (2014), 10, 1036-1044, describes a method that enables probing of structural transitions of proteins in complex biological environments on a large scale by coupling limited proteolysis to generate small peptides amenable to bottom-up proteomic analysis by LC-MS/MS. However, as acknowledged by the authors, the method lacks sufficient sensitivity to access low-abundance proteins and identification of LiP peptides from such low-abundance proteins will require the addition of appropriate sample-enrichment steps.
Lomenich et al., “Identification of Direct Protein Targets of Small Molecules”, ACS Chemical Biology (2010), 6(1), 34-46, describes a method for small-molecule target identification referred to as “DARTS” (Drug Affinity Responsive Target Stability). The method relies on drug-protein interactions based on the idea that the presence of a drug stabilizes the structure of its target protein, which results in increased protease resistance. In the DARTS method protein lysates are incubated with the native drug and then treated with proteases. The target proteins are negatively enriched due to their drug-induced protease resistance while non-target proteins fall victim to proteolysis. However, the method generally lacks sufficient sensitivity for the target protein which could either be insufficiently abundant or its enrichment could be masked by other proteins in the sample.